Synergistic antioxidant combination of delta tocols and polyphenols

ABSTRACT

A source of δ-tocotrienol and/or-tocopherol acts synergistically with an antioxidant source comprising polyphenols, to effect suppression of LDL oxidation in serum. The combination of these two antioxidant sources has wide-ranging applications in treatment of medical conditions arising from free radical generation, including arteriosclerosis and cancer.

FIELD OF THE INVENTION

The present invention relates to compositions comprising a combinationof at least one source of δ-tocols (tocotrienol and/or tocopherol), withan antioxidant source comprising polyphenolic compounds. Due tosynergistic effects, such compositions are useful in treating orpreventing disorders caused by free radicals, such as those relating tocoronary heart disease, cancer and rheumatism.

BACKGROUND OF THE INVENTION

There is ample evidence suggesting that diets rich in fruit andvegetables protect against the development of chronic illnesses such asCoronary Heart Disease (CHD) and cancer, which are the major killerdiseases among affluent populations. It has been recognized for sometime that these beneficial effects are due, at least in part, to theantioxidant components of these foodstuffs, which can inhibit cellulardamage by free radicals. In the body, free radical damage arises due toreactive oxygen species (ROS) being formed endogenously within thetissues, and also as a result of exposure to radicals arising fromcigarette smoke, ionising radiation, air pollution and otherenvironmental insults. These free radicals have the potential tointeract with biological molecules such as proteins, lipids and DNA, andthey are implicated in the aetiology of diverse diseases.

Antioxidants are likely to come into play in many aspects ofmulti-factorial disease conditions such as cancer or CHD, so it isdifficult to establish how their primary effects are exerted. However,in the case of the process of atherogenesis a clear link has beendemonstrated between oxidation of low density lipoprotein (LDL) and theformation of occlusive plaque in the arteries. Vitamin E (α-tocopherol)consumed as part of the diet is naturally present in the blood, where itacts to spare the LDL from oxidation. In vitro experiments haveestablished that other natural sources of antioxidants, such as greentea, can also significantly retard the oxidation of LDL. In vivo studieshave confirmed the in vitro tests, showing that increasing dietaryintakes of some antioxidants, such as Vitamin E, can increase theresistance of LDL to oxidation and thereby reduce lesions in thearteries.

The main antioxidants from dietary sources are vitamins E (α-tocopherol)and C (ascorbic acid), the carotenoids, and polyphenols. Due to anincrease in public awareness of the importance of these micronutrients,it has become commonplace for people to supplement their diets withnatural or synthetic antioxidant sources. However, in spite of thisgeneral shift in favour of healthier eating patterns the incidence ofCHD and cancer continues to be concerning.

It is clearly easier to encourage the use of dietary antioxidantsupplements than to induce radical changes in the diet. But dietarysupplements have their own drawbacks, particularly since there aredetrimental side effects associated with high doses. Recently, forinstance, the US Institute of Medicine advised that the upper limit ofintake of vitamin E should be set at 1000 mg α-tocopherol per day,because of the risk of stroke and uncontrolled bleeding due to theanti-coagulant properties of this nutrient. β-carotene has actually beenlinked to an increase in the incidence of cancer. Thus, at the maximumsafe dosage the antioxidant effect may be less than optimal.

Consequently, there is a need for new antioxidant products, which can beprovided in the form of a food or as a dietary supplement, and which arepotent in preventing CHD, cancer and other diseases associated with freeradical generation, at dosages which do not pose health risks.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided acomposition comprising a mixture of:

-   (a) a source of δ-tocol comprising greater than 2% by weight    δ-tocol; and-   (b) polyphenols,    with the proviso that when (b) comprises citrus flavonoids, (a)    comprises δ-tocopherol.

In some embodiments, the composition may further comprise α-tocopherol,wherein a weight ratio of δ-tocol to α-tocopherol is greater than 0.65.

In a second aspect of the invention there is provided a compositioncomprising in a daily dosage:

-   (a) at least 5 mg δ-tocopherol or of a mixture of δ-tocotrienol and    δ-tocopherol; and-   (b) polyphenols.

In another aspect of the invention there is provided a compositioncomprising in a daily dosage:

-   (a) at least 5 mg of δ-tocotrienol; and-   (b) polyphenols other than citrus flavonoids.

In a further aspect of the invention there is provided a food orbeverage product comprising:

-   (a) a source of δ-tocol comprising greater than 2% by weight    δ-tocol; and-   (b) an antioxidant source comprising polyphenols,    and optionally one or more of carbohydrate, fat, fibre and protein.

In yet another aspect of the invention there is provided apharmaceutical composition or dietary supplement comprising thecomposition of the invention as claimed, with a pharmaceuticallyacceptable carrier.

In another aspect of the invention there is provided use of acomposition according to the invention as claimed, as a scavenger offree radicals.

In a further aspect of the invention there is provided use of acomposition comprising a mixture of:

-   (a) a source of δ-tocol preferably comprising greater than 2% by    weight δ-tocol; and-   (b) an antioxidant source comprising polyphenols,    as a preservative for preventing spoilage of food and beverage    products.

In a further aspect of the invention there is provided a compositionaccording to the invention as claimed, for use as a medicament.

In yet another aspect of this invention there is provided use of acomposition according to the invention as claimed, in the manufacture ofa medicament or nutritional formulation for the prevention or treatmentof a disease condition or corporal damage associated with the generationof free radicals.

In another aspect of the invention there is provided a kit comprising:

-   (a) a source of δ-tocol preferably comprising greater than 2% by    weight δ-tocol; and-   (b) an antioxidant source comprising polyphenols,    for separate, sequential or simultaneous administration.

In a further aspect of the invention there is provided a cosmeticcomposition comprising:

-   (a) a source of δ-tocol preferably comprising greater than 2% by    weight δ-tocol; and-   (b) an antioxidant source comprising polyphenols; and-   (c) a cosmetic base.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating the effects of TRF from palm oil (S;Sugro), green tea extract (GT), and grape seed extract (GS), andcombinations thereof, on the lag phase in an in vitro LDL oxidationassay.

FIG. 2 is a graph showing synergism in the LDL oxidation assay atdifferent concentration ratios of green tea extract (GT):TRF (Fuji).

FIG. 3 is a graph comparing the antioxidant effects of pureδ-tocotrienol and δ-tocopherol in combination with green tea extract(GT) in the LDL oxidation assay.

FIG. 4 compares the antioxidant effects in the LDL oxidation assay ofgreen tea (GT) and Pu Erh red tea (RT) when tested in conjunction withδ-tocotrienol (δ-T).

FIG. 5 demonstrates the effects of using pure epicatechin as asynergistic partner with δ-tocotrienol in the LDL oxidation assay.

DETAILED DESCRIPTION OF THE INVENTION

It has surprisingly been found that the antioxidant properties of acombination of a tocol source comprising δ-tocotrienol and/or-tocopherol, with an antioxidant source comprising polyphenols, are morethan additive, i.e. we have observed a synergistic effect in preventingoxidation in an in vitro LDL oxidation model (which is a representativemodel for free radical oxidation in vivo in general, and peroxidation oflipids in particular).

As a result of this synergy, it is possible to obtain an equivalent orimproved antioxidant effect at lower dosages compared with conventionalantioxidant products. This has the advantage that a powerful antioxidanteffect can be achieved in vivo while avoiding the need to ingest anysingle antioxidant at levels that could threaten health. In addition,the maximum antioxidative benefit obtainable is superior to the maximumbenefit obtainable through use of these antioxidants separately.

Tocopherols and tocotrienols (sometimes collectively known as vitamin E,or “tocols”) are fat-soluble biological membrane components that arestructurally-related, having the same aromatic chromanol “head”. Whereastocopherols have a saturated isoprenoid side chain, tocotrienol sidechains are unsaturated. Tocols are found in α (alpha), β (beta), γ(gamma) and δ (delta) forms, classified according to the position ofsubstitution on the phenol ring. In the context of the present inventionthe generic term “tocol” is used to refer to any isomer of tocotrienolor tocopherol or salt form thereof, or any mixture (racemic orotherwise) of such molecules. “δ-tocols” are a sub-group of thesetocols, i.e. those having a δ-monomethyl group on the benzene ring ofthe chroman moiety, and this term is intended to refer to any isomer ofδ-tocotrienol or δ-tocopherol or salt form thereof, or any mixture(racemic or otherwise) of such molecules. Chemical derivatives andhomologues of tocols are also acceptable for use in the invention,including tocol esters such as the acetate, succinate and palmitate.

The compositions of the invention comprise at least one source (a) ofδ-tocotrienol and/or δ-tocopherol, and are preferably rich in theseδ-tocols, the δ-tocotrienol content or δ-tocopherol content or δ-tocolcontent of the δtocol source preferably being at least 0.1% by weight ofthe source, preferably at least 1%, more preferably greater than 2%,most preferably at least 7%, and optionally at least 9%, by weight ofthe source.

In another embodiment of the invention the δ-tocol content of theδ-tocol source (a) is at least 25% by weight, and preferably at least50% by weight of that source. Optionally the tocol component of thecomposition consists essentially of (e.g. at least 90%, preferably atleast 95% by weight of), or consists of, δ-tocotrienol and/orδ-tocopherol. Individual or mixed δ-tocols can be obtained insubstantially pure form (e.g. ≧90% purity, preferably ≧95% purity) bychemical synthesis or by purification from natural sources, according toconventional procedures.

Tocol sources which are especially rich in δ-tocols include cereals(barley, oats, rice, wheat, rye, amaranths) and plant oils, such as ricebran oil, barley oil, olive oil and palm oil. The source may be enrichedin δ-tocols by conventional processing means, such as that disclosed inWO 91/17985. Preferred sources of δ-tocols for use in the invention arethe Tocotrienol Rich Fractions (TRF; e.g. Nutriene®, TocoTab P40®,Gold-TriE®, Palmvitee®,), obtained by molecular distillation from any ofthe above-named plant oils. Normally, the δ-tocol source, which may be afraction or extract, will comprise a mixture of fat-soluble molecules,including a range of tocopherols and tocotrienols. Optimally, the totaltocol content of the tocol source is at least 30% by weight, preferablyat least 50% by weight, and most preferably at least 70% by weight ofthe source. In order to reach this concentration of tocols it may benecessary to further concentrate commercially available tocol productssuch as TRF.

In one embodiment of the invention the weight ratio of totaltocotrienols to α-tocopherol in the composition is greater than 0.5,preferably greater than 1.0, or even greater than 2.0 or 3.0.Optionally, the composition is substantially free of α-tocopherol,meaning that less than 5%, preferably less than 2% by weight of thetotal tocol content is α-tocopherol.

In a particularly preferred embodiment, source (a) is selected on thebasis that the weight ratio of δ-tocol to α-tocopherol is greater than0.65, preferably at least 1.0. One such source is TocoTab P40® (Fuji), aTRF prepared from palm oil by a process which selectively enriches inδ-tocotrienol. As an example of a δ-tocol source with a very high ratioof δ-tocol to α-tocopherol (>1.5), Covi-Ox™T-30 P (a mixture oftocopherols sold by Cognis GmbH), may be mentioned. By selecting source(a) to have a high ratio of δ-tocol to α-tocopherol it is possible toingest a relatively large dose of antioxidant δ-tocols without the riskof exceeding the maximum daily intake of α-tocopherol recommended bynational health authorities.

Suitable daily doses of total tocol in the composition of the inventionare up to 2000 mg, preferably 5 to 1000 mg, and optimally in the rangeof 50 to 500 mg, generally 100-300 mg. In a unit dosage, the compositionof the invention preferably comprises 0.1 to 50 mg, more preferably 0.25to 20 mg, and most preferably 0.5 to 15 mg δ-tocol. The preferred dailydosages of δ-tocol are up to about 1000 mg, optionally 0.1 to 500 mg,more preferably 0.25 to 125 mg, usually 0.5 to 60 mg, and optionally 5to 25 mg. Where TRF is used as the source (a) of δ-tocol it ispreferably consumed in an amount of 10 to 500 mg/day, more preferably50-250 mg/day, and most preferably 20-100 mg/day. In terms of bodyweight, an appropriate daily dosage range is about 5 μg to 5 mgδ-tocol/kg, especially about 0.1 to 2 mg δ-tocol/kg. The daily dosagemay correspond to a single unit dosage, or may be provided throughmultiple unit dosages.

Although remarkable synergistic effects are observed with each ofδ-tocotrienol and δ-tocopherol, the former performs marginally better inin vitro LDL oxidation studies, and therefore can be administered inlower doses to achieve the same effect. Therefore an optimal compositionaccording to the invention will comprise &tocotrienol, in the presenceor absence of other tocols.

Antioxidant source (b) of the composition of the invention can bedefined as comprising at least one source or substance comprisingpolyphenolic compounds. These polyphenolics contribute to theexceptional antioxidant activities of sources (a) and (b) when used incombination.

In antioxidant source (b) there may be a single polyphenolic source ormixtures of different polyphenolic sources. The term “polyphenol” can bedefined as a compound which possesses an aromatic ring bearing one ormore hydroxy substituents, including functional derivatives. Antioxidantagents containing polyphenols include plant, algal or fungal extracts orfractions rich in polyphenols, including flavonoids (isoflavones,anthocyanins, proanthocyanidins and anthocyanidins, flavans, flavonols,flavones and flavanones). Specific examples of bioflavonoids arecatechins (catechin, epicatechin, gallocatechin, epigallocatechin,epicatechin gallate, epigallocatechin gallate), quercetin, rutin,hesperidin and genistein.

Chemically synthesized or purified polyphenols and mixtures thereof maybe used in place of plant extracts. Polyphenols may be synthesized orextracted from natural sources by any suitable method known to thoseskilled in the art, particularly using food-grade solvents. Liquid andsolid (e.g. granulate or powder form) extracts are suitable.

As a rule, the greater the content of polyphenols in the extract thelarger the synergistic effect observed. Therefore in one embodiment ofthe invention the antioxidant source (b) used in the invention is richin polyphenols, i.e. having a polyphenol content of at least 50% by dryweight, preferably at least 65% by dry weight, and optionally at least75% by dry weight of source (b). Antioxidant source (b) may consistessentially of polyphenols.

In particular, herbal infusions, teas, tisanes and extracts (e.g.lyophilised extracts) made from leaves of various sorts are acclaimedfor their polyphenol-associated free-radical scavenging effects. Herbalsources from which antioxidant preparations can be made includematerials obtained from: tea (Camellia sinensis and Camellia assaimica),rooibos, honeybush, Ginkgo biloba, Phyllanthus species, Catechu gambir,Urtica species, rosemary, sage, mint, etc. Camellia sinensis leaves areespecially preferred. Green tea is particularly potent, as is red tea,white tea and Mohnai tea, but black tea, Oolong tea, yellow tea andjasmine tea may also be used. All varieties of Pu Erh teas and greenteas are preferred. Where the source of polyphenols is leaf material,this material may be freshly-gathered leaves, fresh leaves that aredried immediately after gathering, fresh tea leaves that have been heattreated before drying to inactivate any enzymes present, unfermentedtea, fermented tea, instant tea, tea solids, partially fermented tealeaves, and hot or cold aqueous and alcoholic extracts (tinctures) ofthese leaves, and mixtures thereof.

Polyphenols naturally occurring in Camellia plants and extractstherefrom are especially preferred for use as component (b) in thecompositions of the invention. Included in this group of polyphenols arethe catechins, namely epicatechin, epigallocatechin, epicatechingallate, and epigallocatechin gallate. Sugar salts, sugar esters andother edible physiologically available derivatives of catechins maysubstitute for the naturally-occurring forms.

Effective extracts (e.g. aqueous or alcoholic) as a source ofantioxidant (b) can be obtained from plant parts including leaves(teas), raw or cooked whole fruit, berries and vegetables, nuts (e.g.kola nuts) the skins of fruit, fruit flesh (e.g. orange, prune,tangerine, grapefruit, grape), fruit rind (e.g. citrus fruits), peel,pips, cones (e.g. hops), seeds (e.g. cocoa beans; coffee beans; Silybummarianum) or stones, bark, buds (e.g. Syzygium aromaticum), flowers(e.g. cruciferous vegetables), roots, rhizomes and tubers (e.g. Curcumalonga, Taraxacum officinale, Arctium lappa, Glycyrrhiza glabra, Zingiberofficinalis), stalks, and so on. Grape seed extract is one preferredantioxidant-rich source (b), being rich in proanthocyanidins. Grape skinand grape juice (or red wine extract), maritime pine bark, apples,plums, cherries, red cabbage, wolfberry, hawthorn berry, bilberry,huckleberry, cranberry, elderberry and blueberry and other fruit andvegetables with red, blue or purple pigmentation are other sources ofproanthocyanidins. Vegetables which are rich in polyphenols includepeppers (e.g. chilli peppers), spinach, broccoli, brussels sprouts,cabbage, kale, radishes, turnip and watercress. Extracts made fromfruits and vegetables may have the advantage of providing bothwater-soluble vitamins (C and B complex) and polyphenols.

Interestingly, when grape seed extract and green tea extract were usedin combination as antioxidant component (b) of the composition, thesynergy with δ-tocols was reduced, when compared with green tea extractalone. Therefore, for optimal efficacy it is preferred that when greentea or extracts thereof are used as antioxidant source (b), extractsfrom any part of the grape fruit are omitted from the composition, andvice versa. Diminution of the synergistic effect may be a commonoccurrence when different sources of polyphenols are used incombination. Therefore, in general it is preferred that a singlepolyphenolic antioxidant source is present in the composition.

A unit dosage of the composition of the invention preferably comprises 1mg to 500 mg polyphenols as antioxidant (b), in pure form or in anextract. Daily doses are suitably at least 1 mg, preferably 10 mg to2000 mg, more preferably 100 mg to 1000 mg, and most preferably 250 mgto 700 mg.

In the composition of the invention the polyphenolic antioxidant source(b) is ideally present in equal weight (or concentration) or in weightexcess (concentration excess) relative to the total tocol content of thecomposition, for example in a weight ratio of at least 1:1, preferablyat least 1.5:1, more preferably at least 3:1, and most preferably atleast 6:1.

Preferably the dry weight ratio of polyphenols to δ-tocols in thecomposition of the invention is in the range 1:1 to 200:1, morepreferably 10:1 to 100:1, and most preferably 25:1 to 50:1.

In a preferred embodiment, the composition of the invention comprises asource of δ-tocol(s), in combination with one or more tea catechins, ortea (e.g. green tea or Pu Erh tea) or extracts thereof or mixturesthereof as antioxidant source (b). An appropriate source of δ-tocol(s)is a mix of concentrated tocopherols, such as that sold under the nameof Covi-Ox™T-30 P (Cognis GmbH).

When δ-tocotrienol is the sole δ-tocol in the composition of theinvention, antioxidant source (b) may comprise citrus flavonoids, butwill in addition comprise other polyphenol(s).

Without wishing to be bound by theory, one possible explanation for thesynergy observed using the compositions of this invention is that thereis a cascade of redox cycles occurring in the serum. According to thistheory, oxidation of endogenous α-tocopherol in the LDL complexes sparesoxidation of LDL. Other tocols, especially δ-tocols, regenerateα-tocopherol at the expense of being oxidised themselves. Oxidisedtocols may be recycled by polyphenolic antioxidants or theirmetabolites, and only when these antioxidants are exhausted, after anextended lag phase, does irreversible LDL oxidation start to occur.

Ascorbic acid, a known water-soluble antioxidant, has been postulated torecycle oxidized α-tocopherol in the LDL complexes. However, whenascorbic acid was tested in our in vitro assay, it was found not to havesynergistic antioxidant properties when used in combination with TRF orpurified δ-tocol. The same lack of synergy was observed withN-acetylcysteine, another well-recognized water-soluble antioxidant, andwith α-lipoic acid, which is a fat-soluble antioxidant. None of thesethree antioxidants is polyphenolic, suggesting that the synergy isrelated to the presence of polyphenols in antioxidant agent (b).

The observation that delta tocols are better synergistic partners thanany of the other tocol isomers suggests that the single methyl group onthe phenol ring of the delta tocols is an important factor in thisactivity. If the recycling theory is correct, the implication is thatthe chemistry of the delta tocols allows them to reduce LDLα-tocopherol, or to be reduced by water-soluble antioxidants, in a moreefficient manner than other tocols, or to undergo more redox cycles.

The antioxidant activity of α-tocopherol is not restricted to preventionof LDL oxidation, and it is generally known as a protective vitamin forlipids, and particularly membrane phospholipids. Therefore, thecomposition of the invention can be used to support and supplementendogenous α-tocopherol antioxidant activity in diverse sites andtissues around the body. The composition can be administered innutritional, pharmaceutical or cosmetic form. Although enteraladministration (especially oral) is preferred, topical application isalso envisaged, whether as a pharmaceutical salve or as a skin lotion orcosmetic.

Preferably, components (a) and (b) are provided together inpharmaceutical form or as a dietary supplement, in combination with apharmaceutically acceptable carrier. As an alternative, each componentcan be provided separately, e.g. in kit form, for separate, simultaneousor sequential administration. The pharmaceuticals or supplements arecompositions for enteral (oral, nasal, or rectal), parenteral or topicaladministration, and can be in liquid or solid form.

Typical formulations will comprise (in % by weight) for example, from0.001% to 100%, preferably about 0.1% to 50% by weight of each of activeingredients (a) and (b). Optionally the only active antioxidantcomponents of the pharmaceutical or dietary supplement are tocol source(a) and antioxidant source (b). In some cases the tocol source (a) andantioxidant source (b) together constitute >10%, preferably >25%, andmore preferably >50% by weight of the supplement;

Pharmaceutical compositions and dietary supplements can be produced inthe form of hard or soft (gel) capsules, tablets, dragees, sachets,powders, lozenges, syrups, liquid suspensions, emulsions and solutionsin convenient dosage forms.

Oral pharmaceutical or dietary supplement forms can be made byconventional compounding procedures known in the pharmaceutical art,that is, by mixing the active substances together with ediblepharmaceutically acceptable solid or liquid carriers and/or excipients,e.g. fillers such as cellulose, lactose, sucrose, mannitol, sorbitol,and calcium phosphates and binders, and binders such as starch, gelatin,tragacanth, methylcellulose and/or polyvinylpyrrolidone (PVP). Optionaladditives include lubricants and flow conditioners (e.g. silicic acid,silicon dioxide, talc, stearic acid, magnesium/calcium stearates,polyethylene glycol (PEG) diluents, disintegrating agents (e.g. starch,carboxymethyl starch, cross-linked PVP, agar, alginic acid andalginates) colouring agents, flavouring agents, and melting agents.Dragée cores are provided with suitable, optionally enteric, coatings,there being used inter alia concentrated sugar solutions which maycontain gum arabic, talc, PVP, PEG and/or titanium dioxide, or coatingsolutions in suitable organic solvents or solvent mixtures or, for thepreparation of enteric coatings, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Dyes or pigments may be added tothe tablets or dragee coatings, for example for identification purposesor to indicate different doses of active ingredient.

Other orally administrable pharmaceutical compositions are hard gelatincapsules and also soft, sealed capsules consisting of gelatin and aplasticiser, such as glycerol or sorbitol. The hard gelatin capsules maycomprise the active ingredient in the form of granules, for example inadmixture with fillers, such as lactose, binders, glidants and, ifdesired, stabilisers. In soft capsules the active ingredients arepreferably dissolved or suspended in suitable liquids, such as fattyoils, paraffin oil or liquid polyethylene glycols; it is likewisepossible to add stabilisers.

Since the composition comprises hydrophilic and hydrophobic compounds aconventional emulsifier or surfactant may be employed to disperse theactive ingredients uniformly within the end-product. Optionally, thehydrophobic tocols can be encapsulated in vesicles or micelles forincorporation into low-fat or water-based formulations. One method ofpreparing spray-dried powders containing tocotrienols for compressionmoulding is described in WO 00/27393 and is incorporated herein byreference. Where fats or oils are used as solvents, the water-solublepolyphenolic antioxidant may need to be treated to ensure dispersion,for example by ensuring the particle size is small, and/or by preparingan emulsion using a surfactant having a HLB(Hydrophilicity-Lipophilicity Balance) below 10.

When the composition of the invention is externally applied forcosmetic, hair care or skin care/skin protective purposes, it can be inthe form of a cream, gel, ointment, solid stick, lotion or a solution,optionally together with emulsifiers. Typical compositions includelotions containing water and/or alcohols and emollients such ashydrocarbon oils and waxes, silicone oils, vegetable, animal or marinefats or oils, glyceride derivatives, fatty acids or fatty acid esters,alcohol or alcohol ethers, lanolin and derivatives, polyhydric alcoholsor esters, wax esters, sterols, phospholipids and the like. Conventionalwater-insoluble or aqueous bases for ointments and creams may be used:e.g. fluid paraffin, petrolatum, wool wax, oleyl oleate, and the like.Cetylstearylalcohol and other emulsifying alcohols may be employed, ascan non-alcohol emulsifiers such as Tween 20, Tween 80, and lecithin. Ifdesired, a compound that reduces penetration of, or absorbs, UVradiation can be included in the composition, some examples being PABA,TiO₂, ZnO₂. A representative topical preparation will contain 0.1 to 20%by weight tocols, and at least an equivalent amount by weight of anantioxidant source (b).

The synergistic compositions of the invention can readily beincorporated into nutritional formulations, typically nutraceuticals orfunctional food and beverage products. For example, a δ-tocol source canbe added to a familiar food product which is naturally rich inpolyphenols, some examples being: green tea, herbal teas, coffee, cocoa,grape juice, elderberry juice, citrus juices, berry juices, red wine,beer, and chocolate. Alternatively, purified polyphenols or a source ofpolyphenols are added to a conventional food or beverage product whichis rich in δ-tocols, such as cooking oils, margarines, whole grains,cereals and wholemeal products. Suitable product formats include yellowfat spreads, salad dressings, muesli, granola, muesli bars, crackers,biscuits, and cookies. Another option is to incorporate sources of bothsynergistic partners in a type of food or beverage which is low inendogenous polyphenols and δ-tocols, such as dairy products (cheese,butter, milk, milk-shakes, yogurt, smoothies, ice-cream, cream) andconfectioneries (candies, chewing-gum, desserts, non-dairy toppings,sorbets, icings etc.). In general, food and beverage products of theinvention will comprise nutritional quantities of one or more ofcarbohydrates, protein, fibre and fat.

The polyphenol content of a finished liquid product (drink) willnormally lie in the range of about 0.01 to 1% by weight of the product,preferably 0.025 to 0.5% by weight. In a solid product (a food orreconstitutable drink powder) the usual range for polyphenol content is0.05% to 5%, preferably 0.1 to 2%, by weight.

Edible or drinkable products of the invention will generally contain0.001 to 0.5%, preferably 0.01% to 0.25% by weight of the finishedproduct of δ-tocol source (a).

The beverage compositions of the present invention may be manufacturedand sold for consumption by the consumer in the form of a syrup, anaqueous concentrate, a dry powder, or (effervescent) granules which arediluted with water to yield a beverage, or as a tablet to be taken withwater.

Food and drink products containing the combination of δ-tocol source (a)and antioxidant source (b) are not only attractive to the consumer fortheir associated health benefits, but also have the advantageouscharacteristic of having a long shelf-life due to their potentantioxidant content. Accordingly, there may be no necessity to addartificial preservatives to the food and drink products of theinvention, or at any rate amounts of these additives may be kept to aminimum. Fat- and oil-containing food products are particularlyvulnerable to spoiling through the development of rancidity, and aretherefore prime targets for inclusion of the synergistic antioxidantcomposition of the invention, optionally in conjunction withα-tocopherol.

If desired, other active ingredients may be combined with thetoco/antioxidant source (b) composition of the invention. Particularlypreferred are any natural ingredient or medicament known to have abeneficial effect in lowering cholesterol levels or in combating heartdisease. It may, however, be advisable to exclude beta-carotene from thecomposition due to concerns about carcinogenicity. Other desirablecomponents of the composition are factors that support or supplementfree radical quenching, such as certain minerals, vitamins andmetabolites.

The edible products of the invention optionally comprise conventionalfood additives, such as any of: emulsifiers, stabilizers, sweeteners,flavourings, colouring agents, preservatives, chelating agents, osmoticagents, acidulants, thickeners, texturisers, and so on.

The composition of the invention may be administered with beneficialeffects to prevent, slow the progression of, or treat any diseaseassociated with free radical damage, especially damage resulting inlipid peroxidation. This category includes CHD, cancer (e.g. sarcomasand carcinomas of the colon, pancreas, breast, ovary, prostate gland,lung etc.), rheumatism and inflammation, hepatitis, alcoholism,Alzheimer's disease, dementia, diabetes, multiple sclerosis, HIV andAIDS, collagen degradation, cataracts, macular degeneration, acceleratedaging, neuropathies, myopathies, ischemia-reperfusion injury,haemorrhagic shock, gum disease, cold sores, psoriasis, eczema,seborrhea. The invention can also be applied to counteract the effectsof formation of reactive oxygen species (ROS), whatever the cause,including that resulting from environmental pollution or irradiation,and from treatment with neoplastic drugs.

In particular, this new form of treatment can be applied to prevent ortreat CHD and its underlying causes. Because of the direct connectionbetween prevention of LDL oxidation and retardation of atherosclerosis,stroke patients and cardiac infarct patients, or those at risk of theseconditions, will especially profit from treatment with the claimedcompositions. Type 2 diabetics have an increased preponderance of smalldense LDL, which predisposes to the oxidation of LDL, and in additionhave lower levels of endogenous α-tocopherol. Therefore this patientgroup is a target group particularly well suited to treatment inaccordance with the present method.

Ingestion of the composition of the invention can be employed as acosmetic treatment method, in particular to counteract the developmentof wrinkles, delay skin aging, and improve the overall appearance ofskin, eyes and hair.

Smokers and sunbathers are particularly vulnerable to cancer and agingof the skin and other tissues as a consequence of free radicalgeneration. To some extent they can compensate by maintaining anelevated intake of antioxidants in their diets, which is easilyachievable by consuming the composition of the present invention.

Exercise is believed to trigger an increase in free radical productionby an increase in oxygen generation in the mitochondrial electrontransport chain or by an increase in metal-catalysed free radicalproduction due to mechanical and morphological damage to muscles. It hasbeen reported that lipid and protein oxidation is a consequence ofextended exercise. The present invention provides a convenient methodfor avoiding or minimizing oxidation damage consequent upon physicalactivity.

In accordance with the present invention, a method of preventing ortreating a medical condition caused by generation of free radicals inthe mammalian body comprises administering, to a person in need of suchtreatment, an effective amount of a combination of (a) a tocol sourcecomprising δ-tocols and (b) an antioxidant source comprisingpolyphenols, wherein δ-tocols preferably constitute greater than 2% byweight of the source (a). According to another aspect of the invention,a method of preventing oxidative tissue damage in a mammal due tophysical activity comprises administration of a tocol source comprisingδ-tocols (preferably greater than 2% by weight δ-tocol) in combinationwith a polyphenol-containing antioxidant source.

EXAMPLES

Oxidation of LDL In Vitro

Plasma was obtained from the blood of healthy male and female volunteersand was frozen. After thawing, the plasma was adjusted to a density of1.057 with solid KBr, and overlaid with 0.15M NaCl (density=1.006 kg/l).Tubes were then centrifuged at 400 000 g at 15° C. for 2 hours. The topfraction (chylomicrons+VLDL) was removed. To isolate the LDL fractionthe remaining plasma was adjusted to a density of 1.063 kg/l with solidKBr and the tube was filled with KBr solution (density=1.063). Aftercentrifugation at 400 000 g at 15° C. for 2 hours, the LDL fractionfloating on the top was collected.

The LDL fraction contained between about 300-700 μg/ml proteins. Thefractions were supplemented with ethanolic solutions containing variousextracts or with the same volume of pure ethanol (control). Oxidationwas initiated by adding freshly-prepared CuSO4 solution (25 μmol/l finalconcentration) at 37° C. The kinetics of the oxidation of lipoproteinwas determined by continuously monitoring the change in absorbance at234 nm (at 37° C.) on a UV/VIS spectrophotometer, which correlates withthe increase in conjugated diene absorbance. The effective concentrationof active agent at which 50% of the response was observed is defined asthe EC₅₀.

The time required for the formation of diene is defined as the lagphase. The longer the lag phase the better the protection of LDL againstoxidation. Since it is believed that LDL oxidation contributes to thedevelopment of atherosclerosis, and correlates with the risk ofdeveloping coronary heart disease (CHD) any factor which delays theoxidation of LDL in the in vitro assay is a strong candidate for use ina medicament, food or dietary supplement for treating or preventingthese medical conditions.

Concentration-response curves for the LDL oxidation assay were plottedseparately for each of the tocol preparations, and for the water-solubleantioxidant preparations (see FIGS. 1 to 5). In each experiment, thetocotrienol content of the tocol preparations (TRF) was standardised to0.05 or 0.1 μg/ml.

As can be seen in FIG. 1, green tea extracts (GT, Greenselect®) andgrape seed extract (GS, Leucoselect®), both supplied by Indena, are atleast equal in antioxidant potency to tocols (in this case palm oil TRF“Gold Tri-E” containing 7 wt % δ-tocotrienol and supplied by Sugro, “S”)in this assay (i.e. they have lower EC₅₀ values). Particularcombinations of these two classes of antioxidants were tested in thesame assay. Additive antioxidant effects would have been indicated by alag time corresponding to the addition product of the lag times of theindividual components. In fact, synergy was observed, i.e. the lag timeachieved with the combination of GT/TRF and GS/TRF was significantlylonger than the sum of the individual lag times.

(Sugro TRF was used at a tocotrienols equivalent concentration of 0.1μg/ml. Green tea and grape seed extracts were used at a concentration of0.4 μg/ml, except in the case where green tea and grape seed were usedtogether, when each was used at a concentration of 0.2 μg/ml.)

The results shown in FIG. 2 demonstrate that synergy between green teaextract (GT) and TRF (Fuji, approx. 9 wt % δ-tocotrienol) isdemonstrable at concentration ratios of 1:1 and higher. In theexperiments Fuji TRF was used at a tocotrienols equivalent concentrationof 0.1 μg/ml, and green tea extract was used at a concentration of 0.1,0.4 or 0.6 μg/ml.

FIG. 3 illustrates that purified δ-tocols, when combined with green teaextract (GT), show synergistic antioxidant interactions in the LDLoxidation assay. Here the synergistic properties of this combinationresult in a striking extension to the lag time as compared with the sumof the individual component lag times. The lag phase observed withpurified δ-tocols was also significantly and consistently lengthened incomparison with that observed using other tocol isomers in combinationwith green tea extract under otherwise identical conditions. δ-tocolswere used at a concentration of 0.05 μg/ml, and green tea at 0.3 μg/ml.

FIG. 4 shows that both green tea (GT) and Pu Erh red tea (RT) arecapable of inducing an extended LDL oxidation lag phase when combinedwith δ-tocotrienol (δ-T). The teas were used at a concentration of 0.3μg/ml, and &tocotrienol was used at a concentration of 0.1 μg/ml in theassay.

In FIG. 5 it is shown that pure catechin (epicatechin from Sigma) cansubstitute for green tea extract as a polyphenolic antioxidant partnerwith δ-tocotrienol in the LDL oxidation assay. δ-tocotrienol was used ata concentration of 0.1 μg/ml and epicatechin was used at 0.3 μg/ml.

1. A composition comprising: (a) a source of δ-tocol comprising greaterthan 2% by weight said δ-tocol; and (b) polyphenols, wherein a dryweight ratio of said polyphenols to said δ-tocol is in the range of 25:1to 50:1, and said δ-tocol comprises δ-tocopherol when said polyphenolscomprises citrus flavonoids, and wherein said composition issubstantially free of α-tocopherol.
 2. A composition according to claim1, which in a unit dosage comprises 0.5 to 50 mg δ-tocol.
 3. Acomposition comprising in a daily dosage: (a) at least 5 mg δ-tocolselected from the group consisting of δ-tocopherol, δ-tocotrienol andmixtures thereof; and (b) polyphenols, wherein a dry weight ratio ofsaid polyphenols to said δ-tocol is in the range of 25:1 to 50:1 , andwherein said composition is substantially free of α-tocopherol.
 4. Acomposition comprising in a daily dosage: (a) at least 5 mg of δ-tocolselected from the group consisting of δ-tocopherol, δ-tocotrienol andmixtures thereof; and (b) polyphenols other than citrus flavonoids,wherein a dry weight ratio of said polyphenols to said δ-tocol is in therange of 25:1 to 50:1, and wherein said composition is substantiallyfree of α-tocopherol.
 5. The composition according to claim 1, furthercomprising a catechin.
 6. The composition according to claim 1, furthercomprising α-tocopherol, wherein a weight ratio of δ-tocol toα-tocopherol is greater than 0.65.
 7. The composition according to claim1, wherein δ-tocol is a Tocotrienol Rich Fraction (TRF) source selectedfrom the group consisting of palm oil or rice bran oil and a blend oftocopherols, and said polyphenols is selected from the group consistingof green tea, Pu Erh tea, extracts thereof and mixtures thereof.
 8. Thecomposition according to claim 1, which is substantially free of tocolsother than δ-tocols.
 9. The composition according to claim 1, wherein anantioxidant property of the composition is greater in the case that saidpolyphenols consist of a single polyphenolic antioxidant source than inthe case that said polyphenols include more than one polyphenolicantioxidant source.
 10. The composition according to claim 1, furthercomprising a nutritionally quantity of a carbohydrate, a fat, a fiber ora protein.
 11. The composition according to claim 1, further apharmaceutically acceptable carrier.
 12. The composition according toclaim 11, further comprising antioxidants consisting essentially of saidδ-tocol and said polyphenols.
 13. A kit comprising: (a) a source ofδ-tocol preferably comprising greater than 2% by weight δ-tocol; and (b)an antioxidant source comprising polyphenols, for separate, sequentialor simultaneous administration, wherein a dry weight ratio of saidpolyphenols to said δ-tocol is in the range of 25:1 to 50:1, whereinsaid δ-tocol is selected from the group consisting of δ-tocopherol,δ-tocotrienol and mixtures thereof, and wherein said kit issubstantially free of α-tocopherol.
 14. The composition according toclaim 1, further a cosmetically acceptable carrier.
 15. A method oftreating a medical condition caused by the generation of free radicalscomprising the step of administering to a person in need of suchtreatment an effective amount of a source of δ-tocol comprising greaterthan 2% by weight said δ-tocol and polyphenols, wherein a dry weightratio of said polyphenols to said δ-tocol is in the range of 25:1 to50:1, and said δ-tocol comprises δ-tocopherol when said polyphenolscomprises citrus flavonoids, and wherein administration is substantiallyfree of α-tocopherol.
 16. The method of claim 15, wherein said medicalcondition is selected from the group consisting of arteriosclerosis,rheumatism and cancer.
 17. The method of claim 15, wherein said medicalcondition is caused by physical activity, sun exposure or smoking.
 18. Amethod of preventing spoilage in a food or beverage product comprisingthe step of using a composition comprising a source of δ-tocolpreferably comprising greater than 2% by weight δ-tocol and anantioxidant source comprising polyphenols as a preservative wherein adry weight ratio of said polyphenols to said δ-tocol is in the range of25:1 to 50:1, wherein said δ-tocol is selected from the group consistingof δ-tocopherol, δ-tocotrienol and mixtures thereof, and wherein saidcomposition is substantially free of α-tocopherol.